As will be appreciated by those skilled in the art, very little is understood about many of the complex interactions of insects and plants. For example, defoliation by soybean loopers triggers systemic acquired resistance to stem canker disease and redcrown rot (Russin et al., 1989; Padgett et al., 1994). Conversely, stem-girdling by threecornered alfalfa hoppers predisposes the same plants to the same diseases (Padgett et al., 1994; Hatcher et al., 1995). Thus, the role of insects in triggering resistance or susceptibility to both insects and phytopathogens is still under investigation.
It has recently been recognized that the oral secretions from some herbivores may trigger the release of plant volatiles that may attract the natural enemies of herbivores. For example, .beta.-glucosidase in the regurgitant of Pieris brassicae caterpillars elicits the release of volatile compounds from cabbage leaves (Mattiacci et al., 1995; Proceedings of the National Academy of Science USA). More recently a glutamine-linolenic acid conjugate named volicitin was isolated from the regurgitant of beet armyworms Spodoptera exigua and found to induce the release of volatiles from corn seedlings (Alborn et al., 1997; Science).
Currently, Monsanto Company (700 Chesterfield Village Parkway, St. Louis, Mo. 63198) has expressed the gene encoding the fungal (Asperhillils niger) glucose oxidase in potatoes to confer disease resistance (Wu et al., 1997; Plant Physiol. 115:427-435). Monsanto postulated that active oxygen species perform multiple functions in plant disease, but their exact role in plant resistance to diseases is not fully understood. Monsanto demonstrated H.sub.2 O.sub.2 -mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In Monsanto's research, they provided evidence that the H.sub.2 O.sub.2 -mediated disease resistance in potatoes was effective against a broad range of plant pathogens. Monsanto also investigated the mechanisms underlying the H.sub.2 O.sub.2 -mediated disease resistance in transgenic potato plants. They report that the constitutively elevated levels of H.sub.2 O.sub.2 induce the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the levels of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced.
In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H.sub.2 O.sub.2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a contributing factor to the H.sub.2 O.sub.2 -mediated disease resistance in transgenic plants.
Monsanto conducted further research and reported that plant defense responses to pathogen infection involve the production of active oxygen species including hydrogen peroxide (H.sub.2 O.sub.2). Monsanto obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H.sub.2 O.sub.2 when glucose is oxidized. H.sub.2 O.sub.2 levels were elevated in both leaf and tuber tissues of these plants.
The transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp. carotovora and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H.sub.2 O.sub.2 because the resistance could be counteracted by exogenously added H.sub.2 O.sub.2 degrading catalase.
The transgenic plants with increased levels of H.sub.2 O.sub.2 also exhibited enhanced resistance to potato blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.
The Salk Institute in La Jolla, Calif. has also used a similar approach to produce disease resistant ricc. The Australian Science Foundation CSIRO scientists have also developed a disease resistant cotton using the same gene.
In addition to the use of glucose oxidase in crops for plant resistance, glucose oxidase has been applied to crops to inhibit foliar pathogens, as is discussed by D. R. Fravel, J. A. Lewis, and J. C. Chittams in "Alginate prill formulations of Talaromyces flavus with organic carriers for biocontrol of Verticillium dahliae," Phytopathology 85:165-168 (1985). U.S. Pat. No. 5,094,951, also discusses the production of glucose oxidase in recombinant systems.
While glucose oxidase may be used with various agricultural applications, it may also be used for other applications. For example, glucose oxidase has been used with various food applications. U.S. Pat. No. 5,085,873 shows a process for the treatment of a non-food product for assuring its microbial decontamination. U.S. Pat. No. 4,996,062 shows a glucose oxidase food treatment and storage method. U.S. Pat. No. 4,990,343 shows an enzyme product and method of improving the properties of dough and the quality of bread. U.S. Pat. No. 4,957,749 shows a process for removing oxygen in foodstuffs and in drinks. U.S. Pat. No. 4, 929,451 shows a process for eliminating disagreeable odor from soya milk. U.S. Pat. No. 4,557,927 shows various food products and processes for producing the same. U.S. Pat. No. 3,804,715 shows a process for preparing sugar containing maltose of high purity. U.S. Pat. No. 3,767,531 shows a preparation of insolubilized enzymes and U.S. Pat. No. 4,675,191 shows a method for production of a low alcoholic wine.
Glucose oxidase may also be used for other applications including biomedical and biochemical. For example, glucose oxidase may be used in the glucose monitoring of blood, urine, etc. as discussed by J. A. Lott and K. Turner in "Evaluation of Trinder's glucose oxidase method for measuring glucose in serum and urine," Clin. Chem. 21 (12):1745-1760 (1975). Glucose oxidase may also be used in enzymatic test strips, such as the ones marketed by Lilly under the tradename TES-TAPE.RTM., to detect glucose in urine. Yet another example is shown in U.S. Pat. No. 5,304,468, which shows areagent test strip and apparatus for determination of blood glucose.
Glucose oxidase may also have other medical uses, such as the development of anticancer and/or antitumor agents as reported by C. F. Nathan and Z. A. Cohn in "Antitumor Effects of Hydrogen Peroxide in Vivo," J. Exp. Med, Vol. 154, 1539-1553 (1981) and by Sanmoszuk M. D. Ehrlich and E. Ramzi in "Preclinical Safety Studies of Glucose Oxidase," J. Pharmacol. Exp. Ther. 266(3):1643-1648 (1993). Also, as reported by P. Heiss, S. Bernatz, G. Bruchelt and R. Senekowitsch-Schmidtke in "Cytotoxic Effect of Immunoconjugate Composed of Glucose Oxidase Coupled to a Chimeric Anti-ganglioside (GD2) Antibody on Spheroids," Anticancer Res. 15(6A):2438-2439 (1995). They report that the therapeutic use of the chimeric anti-ganglioside (GD2) antibody shows some success in the therapy of neuroblastomas and melanoma as shown in various Phase I studies. To enhance the effect, glucose oxidase is coupled to the anti-GD2 antibody to produce H.sub.2 O.sub.2 in the presence of glucose and oxygen. H.sub.2 O.sub.2 easily penetrates the target cells in contrast to the antibody.
Glucose oxidase may also be used for the production of antimicrobial products such as soaps and cremes; for example, Kitchen Cupboard Almond Milk Kitchenhand Creme 2 oz. contains glucose and glucose oxidase. Also, glucose oxidase may be used in synthetic saliva, such as Biotene and the like, since many saliva contain an optimum concentration of a natural enzyme system that regulates the microbiological oral ecosystem (glucose oxidase+lactoperoxidase system).
Biochemical applications could also include Immunochemistry. Glucose oxidase may be used for immunohistochemistry, ELISA's and blot detection, such as in the antigen detection system marketed by Vector Laboratories under the tradename VECTASTAIN.RTM. ABC. It could also be used for identifying and/or tracking proteins as reported by J. J. Marchalonis in "Enzymatic Iodination of Proteins," Biochemical Journal, 113, 229-305, (1969) and by J. I. Thorell and B. G. Johansson, Biochemica et Biophysica Acta, 251,363-9, (1969).
Other uses could include enzymatically amplified sensors for amperometry and voltammetry including electrodes designed for amperometric detection of glucose. For example, enzyme reactions have been widely explored in combination with the electrode chemical techniques to add specificity to voltammetry and amperometry. Such strategies are often referred to as "biosensors" since they employ a biomolecule (e g. enzyme, antibody) and can be used for sensing purposes. The most common situation is to use an oxidase enzyme to detect its primary substructrate (e.g. glucose oxidase to detect glucose). The enzyme typically oxidizes the substrate and then transfers reducing equivalence (electrons) to a small molecule (acceptor or mediator) which can be oxidized at the electrode surface. Electrodes designed for the amperometric detection of glucose, lactate and cholesterol are common examples which have used this technique.
Research has been conducted to design various different types of enzyme electrodes. Using the analyte molecule functioning as a mediator, a saturating excess of the enzyme's substrate is used to make the reduced enzyme kenitically inexhaustible. Once an analyte molecule is oxidized at an electrode surface, it is rapidly reduced by the enzyme and is hence available for re-oxidation. This means that each analyte molecule is detected several times on the experimental time scale, thus, the analytical signal is chemically amplified by the enzyme reaction. For example, catechol analytes using glucose oxidase have been proposed.
Thus, a need exists to continue investigating the interaction of insects with plants to explore methods for improving agriculture. There is also a continuing demand for alternative sources of glucose oxidase for various fields including biomedical, biochemical, food production and preservation, and the like.